An LC-MS/MS method was developed and validated for the determination of deoxynivalenol in wheat dust. Extraction was carried out with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. Analysis was performed using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated according to the criteria mentioned in Commission Decision 2002/657/EC. Due to a high contamination level of wheat dust compared to wheat, limit of detection and limit of quantitation levels of 358 ng/g and 717 ng/g, respectively, were obtained. A small survey was executed on raw wheat materials and their corresponding dust samples (n = 12). The samples were analyzed according to the developed procedure. A linear correlation (R² = 0.941) was found for the deoxynivalenol concentration in dust versus the deoxynivalenol concentration in wheat. Therefore, it would be possible to estimate the cereal contamination through dust contamination.